This invention relates to the detection of specific sequences of nucleotides in a variety of nucleic acid samples, and more particularly to those which contain a sequence characterized by a difference in a single base pair from a standard sequence.
In recent years it has been found that many human diseases can be traced directly to genetic mutations. Some commonly known examples include cystic fibrosis, muscular dystrophy, Tay-Sachs disease, hemophilias, phenylketonuria and sickle-cell anemia. Of the over 500 recognized genetic diseases, many can be traced to single base pair mutations.
Two important techniques have been developed in the art for directly detecting these single base pair mutations. However, neither of these approaches can be easily automated. An automated technique is desirable since it has the potential to decrease labor time, decrease the level of skill required, and should increase reliability. In the first of these prior art techniques, the presence or absence of the mutation in a subject is detected by analysis of a restriction digest of the subject's DNA using Southern blotting. (E. Southern, "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis," Journal of Molecular Biology, 98, (1975), 503). For example, sickle-cell disease results from a mutation that changes a glutamic acid residue, coded by the triplet GAG, for a valine residue, coded by GTG, at position 6 in the .beta.-globin chain of hemoglobin. In the mutation of A to T in the base sequence of the .beta.-globin gene, a restriction site for the enzyme MstII (as well as sites for other restriction enzymes) is eliminated. The sickle hemoglobin mutation can therefore be detected by digesting sickle-cell and normal DNA with MstII and using Southern blotting to distinguish the restriction fragments. Normal DNA will generate an MstII fragment 1.1 kilobases long whereas sickle-cell DNA will generate a fragment 1.3 kilobases long.
The specifics of the Southern blot technique are as follows. First, the sample DNA is cut with a restriction enzyme (in this case MstII), and the resultant fragments are separated, based on their size, typically by agarose gel electrophoresis. The gel is then laid onto a piece of nitrocellulose, and a flow of an appropriate buffer is set up through the gel, perpendicular to the direction of electrophoresis, toward the nitrocellulose filter. The flow causes the DNA fragments to be carried out of the gel onto the filter, where they bind, so that the distribution of the DNA fragments in the gel is replicated on the nitrocellulose. The DNA is then denatured and fixed onto the filter. A radioactively labeled probe, complementary to the sequence under study, is then hybridized to the filter, the probe hybridizing to the specific fragment containing the sequence under study. Autoradiography of the nitrocellulose filter then identifies which fragment or fragments contain the sequence under study, each fragment being identified according to its molecular weight. A variation on this technique is to hybridize and do autoradiography directly in the gel, rather than on a nitrocellulose filter. Also, other restriction enzymes may be used provided one of the resulting fragments contains the mutation site of interest.
This direct Southern blot approach used for sickle-cell disease cannot be used, however, for genetic diseases where the mutation does not alter a restriction site, for example, as in .alpha..sub.1 -antitrypsin deficiency, a genetic disease which subjects the individual to greatly increased risk of developing pulmonary emphysema or infantile liver cirrhosis. There, the mutant gene has a single base change (G.fwdarw.A) that leads to an amino acid substitution (GLU.fwdarw.LYS) at residue 342, thereby producing a non-functional protein. This substitution does not, however, create or destroy a restriction site for any of the currently known restriction enzymes as in sickle-cell anemia. Hence, a straightforward analysis of restriction fragments to search for an altered restriction site is not possible. A technique has been developed, however, which can be used in this situation. (See "Detection of Sickle-cell .beta..sup.s -globin Allele by Hybridization with Synthetic Oligonucleotides," by Brenda J. Conner, et al., Proc. Natl. Acad. Sci., Vol 80, pp. 278-282, (Jan 1983), and "Prenatal Diagnosis of .alpha..sub.1 -Antitrypsin Deficiency by Direct Analysis of the Mutation Site in the Gene," by Vincent J. Kidd, et al., New England Journal of Medicine, Vol. 310, No. 10, (March 1984).) This second technique is to synthesize a 19-base long oligonucleotide (hereinafter a 19-mer) that is complementary to the normal .alpha..sub.1 -antitrypsin sequence around the mutation site. The 19-mer is labeled and used as a probe to distinguish normal from mutant genes by raising the stringency of hybridization to a level at which the 19-mer will hybridize stably to the normal gene, to which it is perfectly complementary, but not to the mutant gene, with which it has the single base pair mismatch. (By stringency, it is meant the combination of conditions to which nucleic acids are subject that cause the duplex to dissociate, such as temperature, ionic strength, and concentration of additives, such as formamide. Conditions that are more likely to cause the duplex to dissociate are called "higher" stringency, e.g., higher temperature, lower ionic strength, and higher concentration of formamide.) Similarly, if it is desired to detect the mutant gene, instead of the normal gene, a 19-mer is used which is complementary to the mutant .alpha..sub.1 -antitrypsin sequence around the mutant site. Hence, by using synthetic probes complementary to the sequence of interest in a Southern blot analysis, and varying the stringency, normal and mutant genes can be distinguished.
Although this latter technique is straightforward, it is not without difficulties, especially if an automated procedure is desired. Generally, both the matched and mismatched probes undergo hybridization to the fragment excised by the restriction enzyme, the matched probe being bound at all 19 bases, and the mismatched probe at at most 18 bases. Hence, the relative difference in binding energy between the two probes is quite small. Thus changes in stringency must be delicate enough to dissociate the mismatched probe without also dissociating the matched probe. This would not be a problem with respect to automating the technique were it not for the considerable retention of 19-mer probes by high-molecular weight DNA, presumably due to DNA sequences in the human genome that are somewhat homologous with the synthetic DNA probes used, although this cannot be stated with certainty. This large excess of somewhat homologous sequences in comparison with the .alpha..sub.1 -antitrypsin gene obscures the experimental results and must be treated as background noise in any automated technique and is presently resolved by subjecting the sample to gel electrophoresis and Southern blotting. (See FIGS. 1A and 1B showing the Southern blots for the .alpha..sub.1 -antitrypsin detection scheme reported by Kidd, et al., above.) In this particular instance, this background did not interfere with the diagnosis since a previously developed restriction map indicated that only the band at 2.4 kilobases was relevant. However, it can be seen that most of the probe actually bound is not in the 2.4 kilobase band. In this instance, the time and labor consuming restriction digest and electrophoresis were carried out to separate the DNA sequence of interest, the 2.4 kilobase fragment, from the bulk of the DNA, thereby essentially eliminating background problems.
In most situations involving genetic disease, such restriction maps will already be available, so that the above technique can be quite generally applicable. However, such a technique is not easily automated, just as the previous technique used for sickle-cell disease is not easily automated. What is needed is a technique for detecting single base pair differences between sequences of nucleotides which does not require the use of restriction enzymes, gel electrophoresis, or time consuming autoradiography, and which is readily amenable to automation.